Understanding how and where a particular virulence protein is displayed to the host is critical to understanding the role that protein contributes to pathogenesis. This issue is of central importance for understanding the pathogenesis of any of the myriad serious diseases caused by the Gram- positive pathogen Streptococcus pyogenes (group A streptococcus). What is remarkable is that is that while this bacterium secretes upwards of 100 different polypeptides, it exclusively uses the General Secretory (Sec) pathway for protein export. A key question is how this single pathway can coordinate the secretion and folding of virulence proteins and how these proteins are then trafficked to their final destinations. An important insight into how secretion, folding and trafficking may be coordinated has come from our identification of the ExPortal, a single membrane microdomain that accumulates a high concentration of Sec translocons and accessory biogenesis factors including HtrA and Sortase. Our hypothesis is that the ExPortal functions as an organelle to promote the interaction of nascent unfolded polypeptides secreted by Sec with accessory factors required for folding and biogenesis in order to coordinate these activities with protein trafficking pathways. A more fundamental understanding of secretion and trafficking will require an understanding of the structural basis of ExPortal organization. In this regard, an important advance has been our observation that the ExPortal has characteristics of a membrane microdomain, in that it has an asymmetric lipid content enriched for specific anionic phospholipids. How this asymmetry is maintained, and whether it is necessary for ExPortal function are questions to be addressed in this proposal. Analysis of ExPortal asymmetry will also allow an investigation of how pathways for membrane targeting of presecretory proteins may direct their post-secretory fate, and how the innate immune system, specifically cationic antimicrobial peptides, may directly target the anionic ExPortal to disrupt secretion of surface proteins and toxins. This latter analysis will also serve as the basis for biochemical characterization of the ExPortal and for genetic screens to identify factors required to support ExPortal organization. This analysis may reveal potential drug targets that can be exploited to disrupt the essential processes of protein secretion and trafficking in S. pyogenes and potentially other pathogenic bacteria. )